CN102586291B - Receptor protein kinase gene and expression vector and application thereof - Google Patents
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Abstract
The invention belongs to the field of genetic engineering, and discloses a receptor protein kinase gene and an expression vector and application thereof. The complementary deoxyribonucleic acid (DNA) sequence of the receptor protein kinase gene TaRLK-B is a sequence identifier number 1 (SEQ ID N0.1), and the coded amino acid sequence of the receptor protein kinase gene TaRLK-B is an SEQ ID NO.2. The gene is from a wanghuibai variety of common wheat (Triticum asetivumL.), and is reported in the wheat for the first time. The TaRLK-B is induced by a gibberellic disease in wanghuibai one variety of the wheat capable of resisting the gibberellic disease to be enhanced in expression, and the expression level of the TaRLK-B is much higher than that in Alondra's one variety of the susceptible wheat. The gene is inserted in pAHC25 to obtain an overexpression vector to convert yang wheat 158 one variety of the wheat affected by the gibberellic disease. After identification of T0-generation gibberellic disease resistance, results show that overexpression of the TaRLK-B can improve resistance of varieties of wheat affected by the gibberellic disease on the gibberellic disease.
Description
Technical field
The invention belongs to the genetically engineered field, disclose a receptor protein kinase gene and expression vector and application.
Background technology
The Cereals such as wheat scab (FHB) is a kind of fungal disease, and it can be to wheat, barley and seeding corn and other crops produce disease and directly cause crop failure and quality to descend; On the other hand, the grain of results is owing to polluted by deoxynivalenol (DON) and some other trichothecene toxoid, and humans and animals is eaten by mistake to produce and poisoned.At present along with the warming of global climate, this disease is each wheat planting district rapid spread in the whole world, the genesis of the control wheat scab difficult problem that become international.
Seed selection wheat-resistance to scab kind is the most economical and effective approach of control head blight.Clear and definite its disease-resistant mechanism, clone's disease-resistant related gene is for the control wheat diseases, carry out breeding for disease resistance improvement and have the most important theories directive significance.The disease-resistant wheat molecular mechanism is the hot subject of present wheat scab research.Interaction between head blight pathogenic bacteria and the host's wheat is very complicated.Gibberellic hypha has the characteristic of saprophytic double parasitism, can live the dead volume plant, also can draw nourishment from from the live body phytoparasite; Wheat is non-specialization of microspecies to scab resistance, also is the quantitative inheritance proterties of controlled by multiple genes simultaneously.Expression conditions in analysis of pathogenic bacteria-host's Interaction helps to excavate disease-resistant gene and finds disease-resistant path, and disease-resistant mechanism is significant for illustrating.Utilize various approaches and methods, be subjected to cDNA library, the SSH library of gibberellic hypha abduction delivering such as structure, utilize two-dimensional electrophoresis connexus spectral technology, chip technology etc., screen gene or albumen that some may be relevant with scab resistance, such as various pathogenesis-related proteins, Cytochrome P450, glucanotransferase, abc transport albumen etc., also find and disease-resistant relevant signal path, such as jasmonic approach, ethene approach etc.
Local Wheat Variety Wang-shui-bai is up to the present universally recognized strong to scab resistance, and resistance is stable.This research is subjected to gibberella to induce rear Difference of Gene Expression Profile according to chip detection Wangshuibai and susceptible mutant NAUH117 fringe section, clone a receptor protein kinase gene at Wangshuibai, and with in the sick wheat breed Yangmai No.158 of its conversion sense head blight, in the hope of improving scab resistance.
Summary of the invention
The objective of the invention is the defects for prior art, a kind of receptor protein kinase gene TaRLK-B is provided.
Another object of the present invention provides the expression vector of this gene.
Another purpose of the present invention provides the application of this gene and expression vector.
Purpose of the present invention can be achieved through the following technical solutions:
Receptor protein kinase gene TaRLK-B, from common wheat (Triticum asetivum L.) local variety Wangshuibai, its nucleotides sequence is classified SEQ ID NO.1 as.
The protein TaRLK-B of this receptor kinase gene coded protein, its aminoacid sequence are SEQ ID NO.2.
The expression vector that contains described receptor protein kinase gene TaRLK-B.
The described expression vector that contains receptor protein kinase gene TaRLK-B claimed in claim 1 preferably with pAHC25 for the carrier that sets out, TaRLK-B gene claimed in claim 1 is inserted gained between the Sma I of pAHC25 and Sac I restriction enzyme site.
The application of described receptor protein kinase gene TaRLK-B in making up the wheat-resistance to scab kind.
The described application of expression vector in making up the wheat-resistance to scab kind that contains receptor protein kinase gene TaRLK-B.
Beneficial effect:
The present invention has obtained a receptor protein kinase gene TaRLK-B and coded protein TaRLK-B thereof from the Wheat Species clone first, is reported first in the wheat.Be inserted into expression vector pAHC25, the Overexpression vector of this gene that obtains imports in the susceptible wheat breed, can improve the resistance of sense head blight Scab In Wheat Varieties.TaRLK-B is used for genetic engineering breeding, and it is imported in the susceptible head blight wheat breed, can obtain to possess the wheat germplasm of scab resistance.
Description of drawings
Fig. 1 utilizes China spring third part homology group's nulli-tetrasomes and part 3B deletion of short arm based material that the TaRLK-B gene is carried out the chromosome segment location, and TaRLK-B is navigated to the 0.78-0.87 section 1 of 3BS, Marker; 2, Wangshuibai (Wangshuibai); 3, H117; 4, China spring (Chinese Spring); 5, N3A/T3B; 6, N3A/T3D; 7, N3B/T3D; 8, N3B/T3A; 9, N3D/T3A; 10, N3D/T3B; 11,3BS-3; 12,3BS-8; 13,3BS-9; 14,3BS-1; 15, the water contrast.
Sxemiquantitative RT-PCR X-coordinate in the fringe tissue of Fig. 2 TaRLK-B after Wangshuibai, NAUH117 and Alondra ' s are induced by gibberella represents that gibberella induces the wheatear tissue sample of 0h, 6h, 24h, 48h, 96h
Fig. 3 TaRLK-B overexpression Vector construction
A: plant expression vector pAHC25; The B:pAHC25:TaRLK-B expression vector establishment
Fig. 4 TaRLK-B transforms the T of Yangmai No.158
0PCR Molecular Identification for positive plant
Fig. 5 TaRLK-B transforms the T0 of Yangmai No.158 for the scab resistance qualification result (sick small ear rate) of positive plant
Xj6, xj10, xj26, xj35, xj36, xj37 and the xj38 positive transformed plant for identifying, xj33 and xj34 are for identifying negative plant, and Yangmai No.158 is the transgene receptor wheat, and Wangshuibai is disease-resistant contrast, and Alondra ' s and Mianyang 85-45 are susceptible contrast.
The karyomit(e) synoptic diagram of Fig. 6 Chinese spring and Chinese spring 3B nullisomic/limbs and disappearance system
A, common wheat China spring karyomit(e) synoptic diagram; B, Chinese spring 3B nullisomic/3A limbs karyomit(e) synoptic diagram; C, Chinese spring 3B nullisomic/3D limbs karyomit(e) synoptic diagram; D, Chinese spring 3B disappearance is 3BS-3 karyomit(e) synoptic diagram, 0.87 indication 3BS the short arm of a chromosome disappearance top, 0.13 part; E, Chinese spring 3B disappearance is 3BS-8 karyomit(e) synoptic diagram, 0.78 indication 3BS the short arm of a chromosome disappearance top, 0.22 part
Embodiment
Wangshuibai (Analysis of Combining Ability of the common wheat seed DON content such as Pei Ziyou merchant peak, Acta Agronomica Sinica ACTA AGRONOMICA SINICA, 2007,33 (5): 731-737) be the material of high anti gibberellic disease.This laboratory is in early-stage Study, utilize the fast neutron radiation Wangshuibai, screening obtains sense head blight mutant NAUH117 (Jin xiao, Xinping Jia et al.A Fast-neutron Induced Fragment Deletion of 3BS in wheat variety Wangshuibai Increased Its Susceptibility to Fusarium Head Blight, Chromosome Research, 2011-2-18,19:225-234.), chromosome deletion occurs in the subregion of 3BS in this mutant, and deletion fragment just in time is Wangshuibai anti gibberellic disease main effect QTL region.In order to obtain gene relevant with gibberellic disease resistant in the Wangshuibai, the present invention utilizes the difference expression gene before and after wheat cdna cDNA microarray Wangshuibai and the inoculation of NAUH117 gibberella, therefrom selects important gene to carry out functional verification.
At heading stage small ear is adopted single flower inoculation method inoculation gibberella, the gibberella strain is to separate and cultivation obtains that (gibberella gathers and cultural method is seen: Liu Chuanqin from the field natural occurrence plant, Guan Shuqing, Huang Wei. gibberella saubinetii separation and Culture and inoculation, agricultural modernization, 1998:9), inoculate as negative control totally 4 processing with the water that does not contain gibberella.24h gets the fringe sample after the inoculation, extract respectively total RNA with TRIZOL (invitrogen) by the reagent specification sheets, carry out gene chip hybridization (Affymetrix wheat cdna chip of expression spectrum, part number 900515), this experiment is finished in " Shanghai biochip engineering center of country ".Take experimental group signal and control group signal ratio greater than 2 as standard screening up-regulated expression gene.Wherein 1 gene is speculated as receptor protein kinase analogue (receptor kinase homolog, probe number is Ta.25825.1.A1_at), be subjected to gibberella to induce up-regulated expression in Wangshuibai, up-regulated expression not in NAUH117 is selected and is carried out candidate gene clone research.By Rapid amplification of cDNA ends (RACE), be subjected to gibberella to induce the total length that obtains this gene in the fringe tissue at Wangshuibai.The sequence of this full length gene 1843bp, sequence is shown in SEQ ID NO.1, sequential analysis shows that this sequence comprises a total length ORF, 5 '-UTR (non-translational region) 332bp wherein, 3 '-UTR, 371 bp, ORF (open reading frame) 1140bp, 380 amino acid of encoding, analyze through SMART software (http://smart.embl-heidelberg.de/), this genes encoding a kind of transmembrane protein, have 22 amino acid signal peptide sequences (Signal sequence) at the N end, and comprise the unknown function extracellular region that is formed by about 80 amino acid, the hydrophobic transmembrane zone that middle position is comprised of 28 amino acid; C end comprises that one forms serine/threonine protein kitase (S/TPK) born of the same parents internal area by 211 amino acid, gene with this protein structure is acceptor proteinoid kinases (Receptor-Like Kinase, RLKs), be TaRLK-B with this unnamed gene.
The chromosomal localization of embodiment 2TaRLK-B gene
According to TaRLK-B gene order design special primer P1 (tatttcagcagccctatcac, SEQ ID NO.3) and P2 (tgattgatcctggtagcatt, SEQ ID NO.4), nulli-tetrasomes and the (E.R.Sears of disappearance system with a cover Common Wheat Varieties China spring, Chen Peidu, Weng Yiqun. the history of China spring aneuploid, wheat crops journal, 1990,2:16-19; Rattan far away is grand etc. and common wheat chromosome deletion system breeds, 1995,2:5-8. used nullisomic/limbs and disappearance based material draw (the Wheat Genetics Resource Centre from wheat genetic resource center of the state university of kansas, u.s.a among the present invention, Kansas State University, USA)) genomic dna is that template is carried out pcr amplification reaction, and the TaRLK-B gene is carried out the karyomit(e) physical positioning.PCR program: 10-50ng dna profiling, each 0.5 μ l of the P1 of 10 μ M and P2; 2.5 μ l10 * buffer; 2.5 the dNTP of μ l 2.5mM; 1.5 the Mg of μ l 25mM
2+0.25 μ l (5U/ μ l) Taq polymerase (TaKaRa) adds water to 25 μ l.The PCR reaction conditions is: 94 ℃ of denaturation 3min; 94 ℃ 45 seconds, 48 ℃ 45 seconds, 72 ℃ 1 minute, 33 circulations; 72 ℃ are extended 10min.The PCR product is through 8% polyacrylate hydrogel electrophoresis detection.The difference of amplification banding pattern only appears in this primer in third part homology group nulli-tetrasomes, TaRLK-B is at material (the accompanying drawing 6-b of 3B nullisomic/3A limbs, 3B nullisomic/3D limbs, c) amplification all lacks the band of a 213bp and among the NAUH117, show that TaRLK-B is positioned on the 3B karyomit(e), and lack at NAUH117.Further utilize the (Fig. 6-e of disappearance system of China spring 3B, f) DNA is that template is carried out pcr amplification TaRLK-B is carried out chromosome segment location, with the chromosome segment of this assignment of genes gene mapping in the FL0.78-0.87 of 3BS, namely be positioned at the anti-FHB main effect QTL zone of Wangshuibai 3BS (Fig. 1).
The expression characteristic that embodiment 3TaRLK-B gene is induced by gibberella
Utilize gibberella to Wangshuibai, NAUH117 and sense head blight wheat breed Alondra ' s (Li Minghao, Chen Wei, Xing Liping, etc., the foundation of Common Wheat Varieties Alondra ' s genetic conversion system, Botany Gazette, 2010,45 (4): 466-471.) induce 0h, 6h, 24h, 48h, 96h, use the primer of energy specific amplified TaRLK-B to P1 (tatttcagcagccctatcac, SEQ ID NO.3) and P2 (tgattgatcctggtagcatt, SEQ ID NO.4), induced by gibberella to the TaRLK-B gene and carry out real-time fluorescence quantitative PCR (Q-PCR) analysis.Extract respectively the total RNA of Wangshuibai, NAUH117 and Alondra ' s after inducing different time, reverse transcription synthesizes cDNA the first chain: agents useful for same is all available from Takara company.Add Oligo d (T) 18primers (100nmol/mL) of the total RNA of 1ug, 2ul in the eppendorf of 200ul centrifuge tube, the sterilized water that spends the RNA enzyme is supplied liquor capacity to 10ul; Rotating centrifugal behind the mixing is hatched 10min for 70 ℃ on the PCR instrument, quenching 2min on ice then, centrifugal collection; Add successively following composition on ice: 5 * Reverse Transcriptase XL Buffer 4ul, dNTP Mixture (10mmol/L each) 2ul, Ribonuclease Inhibitor (40U/ul) 0.5 μ l, Reverse Transcriptase XL (AMV) is 1ul (5U/ul), and the sterilized water that spends the RNA enzyme is supplied liquor capacity to 20ul.Place 10min under the mixing, room temperature; On the PCR instrument 42 ℃, 40min; 48 ℃, 30min; 70 ℃, 15min termination reaction, cooled on ice and centrifugal collection; Add 0.5 μ l RNaseH, 37 ℃, 30min; Centrifugal collection saves backup in-20 ℃ after diluting 10 times.
The PCR reaction is in the upper amplification of real-time fluorescence quantitative PCR instrument (MyIQ, Bio-Rad company, the U.S.) and detect fluorescence.Contain 2 * SYBR Green PCR Master Mix 10uL in the 20uLPCR reaction system, 0.5 μ M primer P1 and P2, reverse transcription cDNA template 2uL.Amplification is: 95 ℃ 10 minutes, then 95 ℃ 15 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute, totally 40 circulations.After reaction finishes, carry out the mensuration of melting curve.The gene expression detection level is quantitatively analyzed with the MyiQ system software.The result shows that TaRLK-B is subjected to gibberella to induce up-regulated expression in Wangshuibai, and expression level is the highest behind the 24h, begins downward modulation behind 48h; TaRLK-B disappearance in NAUH117, therefore each period before gibberella is induced and after inducing all can not be detected its expression.In sense head blight wheat breed Alondra ' s, its expression level each time period also all far below the expression amount in Wangshuibai (Fig. 2).The result of Q-PCR shows that TaRLK-B may be relevant with scab resistance.
Embodiment 4TaRLK-B sense expression vector makes up and transforms common wheat Yangmai No.158 and Disease Resistance Identification
Take from the TaRLK-B gene cDNA of Wangshuibai as template, use primer to P3 (tcc
CccgggAtggctgtggtgagtgcctcttt, SEQ ID NO.5) and P4 (c
GagctcTcacctggggcctgatac, SEQ ID NO.6) carry out pcr amplification, reclaim amplified fragments.With Sma I and Sac I amplified production is carried out double digestion, enzyme is cut carrier pAHC25 (Christensen A H after product is inserted into Sma I and Sac I double digestion, Quail P H, Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants, Transgenic Research, 1996,5:213-218.) in, TaRLK-B is placed the multiple clone site place of Ubi promotor back, replace carrier itself with gus gene.Thus target gene TaRLK-B is cloned into the downstream of strong promoter Ubi, obtains expression vector pAHC25:TaRLK-B (Fig. 3).
7 days about 2000 Yangmai No.158 rataria callus of picking preculture, the Overexpression vector pAHC25:TaRLK-B that carries goal gene TaRLK-B is transformed into Yangmai No.158 by particle bombardment, ooze substratum (MS+ABA0.5mg/L+ caseinhydrolysate 500mg/L+2 at height before the bombardment, 4-D2mg/L+ glucose 30g/L+0.4mol/L N.F,USP MANNITOL, pH5.8) upper pre-treatment 4-5 hour, ooze substratum at height after the bombardment and continue to cultivate 16 hours.Afterwards callus is transferred to recovery media (1/2MS+ caseinhydrolysate 500mg/L+2,4-D2mg/L+ sucrose 30g/L, pH5.8) upper dark the cultivation for 2 weeks, transfer them to again (1/2MS+ABA0.5mg/L+ caseinhydrolysate 500mg/L+2 on the screening culture medium that contains weedicide, 4-D1mg/L+ sucrose 30g/L+4mg/L Bialaphos, pH5.8), 2 weeks of screening and culturing.The callus that then will have Herbicid resistant is transferred to (1/2MS+L-paddy ammonia phthalein amine 1mmol/L+ caseinhydrolysate 200mg/L+KT 1mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar 0.8% in the division culture medium, pH5.8) break up, treat to transfer them to when Bud Differentiation grows to 2-4cm root media (1/2MS+KT 1mg/L+ sucrose 30g/L+ agar 0.8%, pH5.8) in.Be about 8cm, root system when healthy and strong to regrowth, can the open pipe hardening 1-2 days, the substratum residue that last flush away root system carries just can be transplanted the engagement alms bowl, obtains totally 40 of regeneration plants.
Extract all regeneration plant genomic dnas, transformed plant is utilized the inner primer P5 of gene (cgacatgacaatgattcac, SEQ ID NO.7) terminator primer P6 (cgctatattttgttttctatcgcgt and on the carrier, SEQ ID NO.8) carries out pcr amplification, to identify positive plant.PCR program: 10-50ng/ul genomic templates, each 0.5 μ l of the P5 of 10 μ M and P6; 2.5 μ l10 * buffer; 2.5 the dNTP of μ l 2.5mM; 1.5 the Mg of μ l 25mM
2+0.25 μ l (5U/ μ l) Taq polymerase (TaKaRa) adds water to 25 μ l.The PCR reaction conditions is: 94 ℃ of denaturations 3 minutes; 94 ℃ 45 seconds, 48 ℃ 45 seconds, 72 ℃ 1 minute, 33 circulations; 72 ℃ were extended 10 minutes.The PCR product is through 8% polyacrylate hydrogel electrophoresis detection.Wherein the 7 strains purpose band of 800bp that can increase is accredited as positive plant, and the strain numbering is followed successively by: xj6, xj10, xj26, xj35, xj36, xj37 and xj38 (Fig. 4).
The positive plant of all evaluations, negative plant (xj33, xj34) are carried out scab resistance identify that (scab resistance is identified the single flower inoculation method that adopts: a Xiao Hua who 5000/ml of 10 μ l gibberella spore suspensions is added drop-wise to the tassel middle part of just blooming, remove sack behind the bagging moisturizing 3d, the sick small ear rate of 21d " Invest, Then Investigate " after the inoculation.Sick small ear rate=(morbidity spikelet number/total spikelet number) * 100%), with unconverted susceptible wheat breed Yangmai No.158, disease-resistant variety Wangshuibai and susceptible variety Alondra ' s, (Pei Ziyou merchant peak etc., Mianyang 8545, the Analysis of Combining Ability of common wheat seed DON content, Acta Agronomica Sinica, ACTA AGRONOMICA SINICA, 2007,33 (5): 731-737) be contrast.Transgenosis T
0Compare gibberellic disease resistant level be improved (Fig. 5) with unconverted Yangmai No.158 for positive plant xj26, xj35, xj36, xj37.
Claims (6)
1. receptor protein kinase gene
TaRLK-B, it is characterized in that nucleotides sequence classifies SEQ ID NO.1 as.
2. the protein of the described receptor protein kinase gene of claim 1 TaRLK-B coding is characterized in that aminoacid sequence is SEQ ID NO.2.
3. contain receptor protein kinase gene claimed in claim 1
TaRLK-BExpression vector.
4. expression vector according to claim 3 is characterized in that the described receptor protein kinase gene claimed in claim 1 that contains
TaRLK-BExpression vector be with
PAHC25Be the carrier that sets out, with claimed in claim 1
TaRLK-BGene inserts
PAHC25 Sma IWith
Sac IGained between restriction enzyme site.
5. receptor protein kinase gene claimed in claim 1
TaRLK-BApplication in making up the wheat-resistance to scab kind.
6. claim 3 or the 4 described receptor protein kinase genes that contain
TaRLK-BThe application of expression vector in making up the wheat-resistance to scab kind.
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